Trypanosoma brucei, the causative agent of African sleeping sickness, is a protozoa whose study has brought to light novel mechanisms in eukaryotic gene expression. These include trans-splicing and editing of RNA, and the phenomena of antigenic variation by which the surface coat, the variant surface glycoprotein (VSG), changes once the host mounts an immune response to the parasite. Switching from one VSG to another occurs either by gene conversion of a previously silent gene copy to an expression site located at the chromosomal telomere, or by activation of an alternative telomeric expression site. It is unknown, though, how this transcriptional activation occurs and the possibility has been raised that VSG expression is accomplished by a unique RNA polymerase. The ultimate objective of the proposed research is to understand the molecular mechanisms which allow for selective and stage specific transcription of VSG genes. Initial studies will focus on identifying which class of polymerase transcribes the VSG genes and to define the VSG transcription initiation site. Since the 5' end of the mature mRNA in trypanosomes is derived from a different transcription unit, a novel approach using U.V. crosslinking will be adapted for the mapping of trypanosome transcription units. RNA polymerase complexes will be crosslinked to the DNA template by U.V. irradiation and immunoprecipitated. Labeled DNA fragments will then be hybridized to the RNA polymerase-DNA template complexes to determine if the test fragment corresponds to a transcriptionally active genomic DNA sequence. In this way regions of VSG transcription initiation will be identified. Furthermore, by use of antibodies specific to each of the RNA polymerases, this methodology will allow identification of which polymerase is associated with VSG transcription. U.V. crosslinking will be further used to determine the transcription initiation region of other cloned- trypanosome genes including tubulin, 5S RNA and 28S RNA. These regions will be sequenced to identify conserved trypanosome promotor elements. To assay for promotor sequences, efforts will be directed towards developing an in vitro transcription system. Once available, such a system will allow dissection of putative promotor sequences by in vitro mutagenesis. The future potential applications of this work lie not only in identifying new sites for possible pharmacologic intervention and vaccine development for African sleeping sickness and related diseases, but also may help in understanding the basic mechanisms of transcription regulation in higher eukaryots. Since trypanosomes appear to have diverged early in eukaryotic evolution, fine analysis of their transcription promotors and regulatory elements may reveal common and unique themes in how genetic information is processed.